Massive parallel method for decoding DNA and RNA

ABSTRACT

This invention provides methods for attaching a nucleic acid to a solid surface and for sequencing nucleic acid by detecting the identity of each nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction. The invention also provides nucleotide analogues which comprise unique labels attached to the nucleotide analogue through a cleavable linker, and a cleavable chemical group to cap the —OH group at the 3′-position of the deoxyribose.

This application is a continuation of U.S. Ser. No. 11/810,509, filedJun. 5, 2007 now U.S. Pat. No. 7,790,869, now allowed, a continuation ofU.S. Ser. No. 10/702,203, filed Nov. 4, 2003, now U.S. Pat. No.7,345,159, issued Mar. 18, 2008, a divisional of U.S. Ser. No.09/972,364, filed Oct. 5, 2001, now U.S. Pat. No. 6,664,079, issued Dec.16, 2003, claiming benefit of U.S. Provisional Application No.60/300,894, filed Jun. 26, 2001, and is a continuation-in-part of U.S.Ser. No. 09/684,670, filed Oct. 6, 2000, now abandoned, the contents ofeach of which are hereby incorporated in their entireties into thisapplication.

The invention disclosed herein was made with government support underNational Science Foundation award no. BES0097793. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced inparentheses by author and year. Full citations for these references maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

The ability to sequence deoxyribonucleic acid (DNA) accurately andrapidly is revolutionizing biology and medicine. The confluence of themassive Human Genome Project is driving an exponential growth in thedevelopment of high throughput genetic analysis technologies. This rapidtechnological development involving chemistry, engineering, biology, andcomputer science makes it possible to move from studying single genes ata time to analyzing and comparing entire genomes.

With the completion of the first entire human genome sequence map, manyareas in the genome that are highly polymorphic in both exons andintrons will be known. The pharmacogenomics challenge is tocomprehensively identify the genes and functional polymorphismsassociated with the variability in drug response (Roses, 2000).Resequencing of polymorphic areas in the genome that are linked todisease development will contribute greatly to the understanding ofdiseases, such as cancer, and therapeutic development. Thus,high-throughput accurate methods for resequencing the highly variableintron/exon regions of the genome are needed in order to explore thefull potential of the complete human genome sequence map. The currentstate-of-the-art technology for high throughput DNA sequencing, such asused for the Human Genome Project (Pennisi 2000), is capillary array DNAsequencers using laser induced fluorescence detection (Smith et al.,1986; Ju et al. 1995, 1996; Kheterpal et al. 1996; Salas-Solano et al.1998). Improvements in the polymerase that lead to uniform terminationefficiency and the introduction of thermostable polymerases have alsosignificantly improved the quality of sequencing data (Tabor andRichardson, 1987, 1995). Although capillary array DNA sequencingtechnology to some extent addresses the throughput and read lengthrequirements of large scale DNA sequencing projects, the throughput andaccuracy required for mutation studies needs to be improved for a widevariety of applications ranging from disease gene discovery to forensicidentification. For example, electrophoresis based DNA sequencingmethods have difficulty detecting heterozygotes unambiguously and arenot 100% accurate in regions rich in nucleotides comprising guanine orcytosine due to compressions (Bowling et al. 1991; Yamakawa et al.1997). In addition, the first few bases after the priming site are oftenmasked by the high fluorescence signal from excess dye-labeled primersor dye-labeled terminators, and are therefore difficult to identify.Therefore, the requirement of electrophoresis for DNA sequencing isstill the bottleneck for high-throughput DNA sequencing and mutationdetection projects.

The concept of sequencing DNA by synthesis without using electrophoresiswas first revealed in 1988 (Hyman, 1988) and involves detecting theidentity of each nucleotide as it is incorporated into the growingstrand of DNA in a polymerase reaction. Such a scheme coupled with thechip format and laser-induced fluorescent detection has the potential tomarkedly increase the throughput of DNA sequencing projects.Consequently, several groups have investigated such a system with an aimto construct an ultra high-throughput DNA sequencing procedure(Cheeseman 1994, Metzker et al. 1994). Thus far, no complete success ofusing such a system to unambiguously sequence DNA has been reported. Thepyrosequencing approach that employs four natural nucleotides(comprising a base of adenine (A), cytosine (C), guanine (G), or thymine(T)) and several other enzymes for sequencing DNA by synthesis is nowwidely used for mutation detection (Ronaghi 1998). In this approach, thedetection is based on the pyrophosphate (PPi) released during the DNApolymerase reaction, the quantitative conversion of pyrophosphate toadenosine triphosphate (ATP) by sulfurylase, and the subsequentproduction of visible light by firefly luciferase. This procedure canonly sequence up to 30 base pairs (bps) of nucleotide sequences, andeach of the 4 nucleotides needs to be added separately and detectedseparately. Long stretches of the same bases cannot be identifiedunambiguously with the pyrosequencing method.

More recent work in the literature exploring DNA sequencing by asynthesis method is mostly focused on designing and synthesizing aphotocleavable chemical moiety that is linked to a fluorescent dye tocap the 3′-OH group of deoxynucleoside triphosphates (dNTPs) (Welch etal. 1999). Limited success for the incorporation of the 3′-modifiednucleotide by DNA polymerase is reported. The reason is that the3′-position on the deoxyribose is very close to the amino acid residuesin the active site of the polymerase, and the polymerase is thereforesensitive to modification in this area of the deoxyribose ring. On theother hand, it is known that modified DNA polymerases (Thermo Sequenaseand Taq FS polymerase) are able to recognize nucleotides with extensivemodifications with bulky groups such as energy transfer dyes at the5-position of the pyrimidines (T and C) and at the 7-position of purines(G and A) (Rosenblum et al. 1997, Zhu et al. 1994). The ternarycomplexes of rat DNA polymerase, a DNA template-primer, anddideoxycytidine triphosphate (ddCTP) have been determined (Pelletier etal. 1994) which supports this fact. As shown in FIG. 1, the 3-Dstructure indicates that the surrounding area of the 3′-position of thedeoxyribose ring in ddCTP is very crowded, while there is ample spacefor modification on the 5-position the cytidine base.

The approach disclosed in the present application is to make nucleotideanalogues by linking a unique label such as a fluorescent dye or a masstag through a cleavable linker to the nucleotide base or an analogue ofthe nucleotide base, such as to the 5-position of the pyrimidines (T andC) and to the 7-position of the purines (G and A), to use a smallcleavable chemical moiety to cap the 3′-OH group of the deoxyribose tomake it nonreactive, and to incorporate the nucleotide analogues intothe growing DNA strand as terminators. Detection of the unique labelwill yield the sequence identity of the nucleotide. Upon removing thelabel and the 3′-OH capping group, the polymerase reaction will proceedto incorporate the next nucleotide analogue and detect the next base.

It is also desirable to use a photocleavable group to cap the 3′-OHgroup. However, a photocleavable group is generally bulky and thus theDNA polymerase will have difficulty to incorporate the nucleotideanalogues containing a photocleavable moiety capping the 3′-OH group. Ifsmall chemical moieties that can be easily cleaved chemically with highyield can be used to cap the 3′-OH group, such nucleotide analoguesshould also be recognized as substrates for DNA polymerase. It has beenreported that 3′-O-methoxy-deoxynucleotides are good substrates forseveral polymerases (Axelrod et al. 1978). 3′-O-allyl-dATP was alsoshown to be incorporated by Ventr(exo-) DNA polymerase in the growingstrand of DNA (Metzker et al. 1994). However, the procedure tochemically cleave the methoxy group is stringent and requires anhydrousconditions. Thus, it is not practical to use a methoxy group to cap the3′-OH group for sequencing DNA by synthesis. An ester group was alsoexplored to cap the 3′-OH group of the nucleotide, but it was shown tobe cleaved by the nucleophiles in the active site in DNA polymerase(Canard et al. 1995). Chemical groups with electrophiles such as ketonegroups are not suitable for protecting the 3′-OH of the nucleotide inenzymatic reactions due to the existence of strong nucleophiles in thepolymerase. It is known that MOM (—CH₂OCH₃) and allyl (—CH₂CH═CH₂)groups can be used to cap an —OH group, and can be cleaved chemicallywith high yield (Ireland et al. 1986; Kamal et al. 1999). The approachdisclosed in the present application is to incorporate nucleotideanalogues, which are labeled with cleavable, unique labels such asfluorescent dyes or mass tags and where the 3′-OH is capped with acleavable chemical moiety such as either a MOM group (—CH₂OCH₃) or anallyl group (—CH₂CH═CH₂), into the growing strand DNA as terminators.The optimized nucleotide set (_(3′-RO)-A-_(LABEL1),_(3′-RO)-C-_(LABEL2), _(3′-RO)-G-_(LABEL3), _(3′-RO)-T-_(LABEL4), whereR denotes the chemical group used to cap the 3′-OH) can then be used forDNA sequencing by the synthesis approach.

There are many advantages of using mass spectrometry (MS) to detectsmall and stable molecules. For example, the mass resolution can be asgood as one dalton. Thus, compared to gel electrophoresis sequencingsystems and the laser induced fluorescence detection approach which haveoverlapping fluorescence emission spectra, leading to heterozygotedetection difficulty, the MS approach disclosed in this applicationproduces very high resolution of sequencing data by detecting thecleaved small mass tags instead of the long DNA fragment. This methodalso produces extremely fast separation in the time scale ofmicroseconds. The high resolution allows accurate digital mutation andheterozygote detection. Another advantage of sequencing with massspectrometry by detecting the small mass tags is that the compressionsassociated with gel based systems are completely eliminated.

In order to maintain a continuous hybridized primer extension productwith the template DNA, a primer that contains a stable loop to form anentity capable of self-priming in a polymerase reaction can be ligatedto the 3′ end of each single stranded DNA template that is immobilizedon a solid surface such as a chip. This approach will solve the problemof washing off the growing extension products in each cycle.

Saxon and Bertozzi (2000) developed an elegant and highly specificcoupling chemistry linking a specific group that contains a phosphinemoiety to an azido group on the surface of a biological cell. In thepresent application, this coupling chemistry is adopted to create asolid surface which is coated with a covalently linked phosphine moiety,and to generate polymerase chain reaction (PCR) products that contain anazido group at the 5′ end for specific coupling of the DNA template withthe solid surface. One example of a solid surface is glass channelswhich have an inner wall with an uneven or porous surface to increasethe surface area. Another example is a chip.

The present application discloses a novel and advantageous system forDNA sequencing by the synthesis approach which employs a stable DNAtemplate, which is able to self prime for the polymerase reaction,covalently linked to a solid surface such as a chip, and 4 uniquenucleotides analogues (_(3′-RO-)A-_(LABEL1), _(3′-RO)-C-_(LABEL2),_(3′-RO)-G-_(LABEL3), _(3′-RO)-T-_(LABEL4)). The success of this novelsystem will allow the development of an ultra high-throughput and highfidelity DNA sequencing system for polymorphism, pharmacogeneticsapplications and for whole genome sequencing. This fast and accurate DNAresequencing system is needed in such fields as detection of singlenucleotide polymorphisms (SNPs) (Chee et al. 1996), serial analysis ofgene expression (SAGE) (Velculescu et al. 1995), identification inforensics, and genetic disease association studies.

SUMMARY OF THE INVENTION

This invention is directed to a method for sequencing a nucleic acid bydetecting the identity of a nucleotide analogue after the nucleotideanalogue is incorporated into a growing strand of DNA in a polymerasereaction, which comprises the following steps:

-   -   (i) attaching a 5′ end of the nucleic acid to a solid surface;    -   (ii) attaching a primer to the nucleic acid attached to the        solid surface;    -   (iii) adding a polymerase and one or more different nucleotide        analogues to the nucleic acid to thereby incorporate, a        nucleotide analogue into the growing strand of DNA, wherein the        incorporated nucleotide analogue terminates the polymerase        reaction and wherein each different nucleotide analogue        comprises (a) a base selected from the group consisting of        adenine, guanine, cytosine, thymine, and uracil, and their        analogues; (b) a unique label attached through a cleavable        linker to the base or to an analogue of the base; (c) a        deoxyribose; and (d) a cleavable chemical group to cap an —OH        group at a 3′-position of the deoxyribose;    -   (iv) washing the solid surface to remove unincorporated        nucleotide analogues;    -   (v) detecting the unique label attached to the nucleotide        analogue that has been incorporated into the growing strand of        DNA, so as to thereby identify the incorporated nucleotide        analogue;    -   (vi) adding one or more chemical compounds to permanently cap        any unreacted —OH group on the primer attached to the nucleic        acid or on a primer extension strand formed by adding one or        more nucleotides or nucleotide analogues to the primer;    -   (vii) cleaving the cleavable linker between the nucleotide        analogue that was incorporated into the growing strand of DNA        and the unique label;    -   (viii) cleaving the cleavable chemical group capping the —OH        group at the 3′-position of the deoxyribose to uncap the —OH        group, and washing the solid surface to remove cleaved        compounds; and    -   (ix) repeating steps (iii) through (viii) so as to detect the        identity of a newly incorporated nucleotide analogue into the        growing strand of DNA;    -   wherein if the unique label is a dye, the order of steps (v)        through (vii) is: (v), (vi), and (vii); and    -   wherein if the unique label is a mass tag, the order of        steps (v) through (vii) is: (vi), (vii), and (v).

The invention provides a method of attaching a nucleic acid to a solidsurface which comprises:

-   -   (i) coating the solid surface with a phosphine moiety,    -   (ii) attaching an azido group to a 5′ end of the nucleic acid,        and    -   (iii) immobilizing the 5′ end of the nucleic acid to the solid        surface through interaction between the phosphine moiety on the        solid surface and the azido group on the 5′ end of the nucleic        acid.

The invention provides a nucleotide analogue which comprises:

-   -   (a) a base selected from the group consisting of adenine or an        analogue of adenine, cytosine or an analogue of cytosine,        guanine or an analogue of guanine, thymine or an analogue of        thymine, and uracil or an analogue of uracil;    -   (b) a unique label attached through a cleavable linker to the        base or to an analogue of the base;    -   (c) a deoxyribose; and    -   (d) a cleavable chemical group to cap an —OH group at a        3′-position of the deoxyribose.

The invention provides a parallel mass spectrometry system, whichcomprises a plurality of atmospheric pressure chemical ionization massspectrometers for parallel analysis of a plurality of samples comprisingmass tags.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The 3D structure of the ternary complexes of rat DNA polymerase,a DNA template-primer, and dideoxycytidine triphosphate (ddCTP). Theleft side of the illustration shows the mechanism for the addition ofddCTP and the right side of the illustration shows the active site ofthe polymerase. Note that the 3′ position of the dideoxyribose ring isvery crowded, while ample space is available at the 5 position of thecytidine base.

FIG. 2A-2B: Scheme of sequencing by the synthesis approach. A: Examplewhere the unique labels are dyes and the solid surface is a chip. B:Example where the unique labels are mass tags and the solid surface ischannels etched into a glass chip. A, C, G, T; nucleotide triphosphatescomprising bases adenine, cytosine, guanine, and thymine; d, deoxy; dd,dideoxy; R, cleavable chemical group used to cap the —OH group; Y,cleavable linker.

FIG. 3: The synthetic scheme for the immobilization of an azido (N₃)labeled DNA fragment to a solid surface coated with a triarylphosphinemoiety. Me, methyl group; P, phosphorus; Ph, phenyl.

FIG. 4: The synthesis of triarylphosphine N-hydroxysuccinimide (NHS)ester.

FIG. 5: The synthetic scheme for attaching an azido (N₃) group through alinker to the 5′ end of a DNA fragment, which is then used to couplewith the triarylphosphine moiety on a solid surface. DMSO,dimethylsulfonyl oxide.

FIG. 6A-6B: Ligate the looped primer (B) to the immobilized singlestranded DNA template forming a self primed DNA template moiety on asolid surface. P (in circle), phosphate.

FIG. 7: Examples of structures of four nucleotide analogues for use inthe sequencing by synthesis approach. Each nucleotide analogue has aunique fluorescent dye attached to the base through a photocleavablelinker and the 3′-OH is either exposed or capped with a MOM group or anallyl group. FAM, 5-carboxyfluorescein; R6G, 6-carboxyrhodamine-6G; TAM,N,N,N′,N′-tetramethyl-6-carboxyrhodamine; ROX, 6-carboxy-X-rhodamine.R═H, CH₂OCH₃ (MOM) or CH₂CH═CH₂ (Allyl).

FIG. 8: A representative scheme for the synthesis of the nucleotideanalogue _(3′-RO)-G-_(Tam). A similar scheme can be used to create theother three modified nucleotides: _(3′-RO)-A-_(Dye1),_(3′-RO)-C-_(Dye2), _(3′-RO)-T-_(Dye4) (i)tetrakis(triphenylphosphine)palladium(0); (ii) POCl₃,Bn₄N⁺pyrophosphate; (iii) NH₄OH; (iv) Na₂CO₃/NaHCO₃ (pH=9.0)/DMSO.

FIG. 9: A scheme for testing the sequencing by synthesis approach. Eachnucleotide, modified by the attachment of a unique fluorescent dye, isadded one by one, based on the complimentary template. The dye isdetected and cleaved to test the approach. Dye1=Fam; Dye2=R6G; Dye3=Tam;Dye4=Rox.

FIG. 10: The expected photocleavage products of DNA containing aphoto-cleavable dye (Tam). Light absorption (300-360 nm) by the aromatic2-nitrobenzyl moiety causes reduction of the 2-nitro group to a nitrosogroup and an oxygen insertion into the carbon-hydrogen bond located inthe 2-position followed by cleavage and decarboxylation (Pillai 1980).

FIG. 11: Synthesis of PC-LC-Biotin-FAM to evaluate the photolysisefficiency of the fluorophore coupled with the photocleavable linker2-nitrobenzyl group.

FIG. 12: Fluorescence spectra (λ_(ex)=480 nm) of PC-LC-Biotin-FAMimmobilized on a microscope glass slide coated with streptavidin (a);after 10 min photolysis (λ_(irr)=350 nm; ˜0.5 mW/cm²) (b); and afterwashing with water to remove the photocleaved dye (c).

FIG. 13A-13B: Synthetic scheme for capping the 3′-OH of nucleotide.

FIG. 14: Chemical cleavage of the MOM group (top row) and the allylgroup (bottom row) to free the 3′-OH in the nucleotide.CITMS=chlorotrimethylsilane.

FIG. 15A-15B: Examples of energy transfer coupled dye systems, where Famor Cy2 is employed as a light absorber (energy transfer donor) andCl₂Fam, Cl₂R6G, Cl₂Tam, or Cl₂Rox as an energy transfer acceptor. Cy2,cyanine; FAM, 5-carboxyfluorescein; R6G, 6-carboxyrhodamine-6G; TAM,N,N,N′,N′-tetramethyl-6-carboxyrhodamine; ROX, 6-carboxy-X-rhodamine.

FIG. 16: The synthesis of a photocleavable energy transfer dye-labelednucleotide. DMF, dimethylformide.DEC=1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride. R═H,CH₂OCH₃ (MOM) or CH₂CH═CH₂ (Allyl).

FIG. 17: Structures of four mass tag precursors and four photoactivemass tags. Precursors: a) acetophenone; b) 3-fluoroacetophenone; c)3,4-difluoroacetophenone; and d) 3,4-dimethoxyacetophenone. Fourphotoactive mass tags are used to code for the identity of each of thefour nucleotides (A, C, G, T).

FIG. 18: Atmospheric. Pressure Chemical Ionization (APCI) mass spectrumof mass tag precursors shown in FIG. 17.

FIG. 19: Examples of structures of four nucleotide analogues for use inthe sequencing by synthesis approach. Each nucleotide analogue has aunique mass tag attached to the base through a photocleavable linker,and the 3′-OH is either exposed or capped with a MOM group or an allylgroup. The square brackets indicated that the mass tag is cleavable.R═H, CH₂OCH₃ (MOM) or CH₂CH═CH₂ (Allyl).

FIG. 20: Example of synthesis of NHS ester of one mass tag (Tag-3). Asimilar scheme is used to create other mass tags.

FIG. 21: A representative scheme for the synthesis of the nucleotideanalogue _(3′-RO)-G-_(Tag3). A similar scheme is used to create theother three modified bases _(3′-RO)-A-_(Tag1), _(3′-RO)-C-_(Tag2),_(3′-RO)-T-_(Tag4) (i) tetrakis(triphenylphosphine)palladium(0); (ii)POCl₃, Bn₄N⁺pyrophosphate; (iii) NH₄OH; (iv) Na₂CO₃/NaHCO₃(pH=9.0)/DMSO.

FIG. 22: Examples of expected photocleavage products of DNA containing aphotocleavable mass tag.

FIG. 23: System for DNA sequencing comprising multiple channels inparallel and multiple mass spectrometers in parallel. The example shows96 channels in a silica glass chip.

FIG. 24: Parallel mass spectrometry system for DNA sequencing. Exampleshows three mass spectrometers in parallel. Samples are injected intothe ion source where they are mixed with a nebulizer gas and ionized. Aturbo pump is used to continuously sweep away free radicals, neutralcompounds and other undesirable elements coming from the ion source. Asecond turbo pump is used to generate a continuous vacuum in all threeanalyzers and detectors simultaneously. The acquired signal is thenconverted to a digital signal by the A/D converter. All three signalsare then sent to the data acquisition processor to convert the signal toidentify the mass tag in the injected sample and thus identify thenucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are presented as an aid in understanding thisinvention.

As used herein, to cap an —OH group means to replace the “H” in the —OHgroup with a chemical group. As disclosed herein, the —OH group of thenucleotide analogue is capped with a cleavable chemical group. To uncapan —OH group means to cleave the chemical group from a capped —OH groupand to replace the chemical group with “H”, i.e., to replace the “R” in—OR with “H” wherein “R” is the chemical group used to cap the —OHgroup.

The nucleotide bases are abbreviated as follows: adenine (A), cytosine(C), guanine (G), thymine (T), and uracil (U).

An analogue of a nucleotide base refers to a structural and functionalderivative of the base of a nucleotide which can be recognized bypolymerase as a substrate. That is, for example, an analogue of adenine(A) should form hydrogen bonds with thymine (T), a C analogue shouldform hydrogen bonds with G, a G analogue should form hydrogen bonds withC, and a T analogue should form hydrogen bonds with A, in a double helixformat. Examples of analogues of nucleotide bases include, but are notlimited to, 7-deaza-adenine and 7-deaza-guanine, wherein the nitrogenatom at the 7-position of adenine or guanine is substituted with acarbon atom.

A nucleotide analogue refers to a chemical compound that is structurallyand functionally similar to the nucleotide, i.e. the nucleotide analoguecan be recognized by polymerase as a substrate. That is, for example, anucleotide analogue comprising adenine or an analogue of adenine shouldform hydrogen bonds with thymine, a nucleotide analogue comprising C oran analogue of C should form hydrogen bonds with G, a nucleotideanalogue comprising G or an analogue of G should form hydrogen bondswith C, and a nucleotide analogue comprising T or an analogue of Tshould form hydrogen bonds with A, in a double helix format. Examples ofnucleotide analogues disclosed herein include analogues which comprisean analogue of the nucleotide base such as 7-deaza-adenine or7-deaza-guanine, wherein the nitrogen atom at the 7-position of adenineor guanine is substituted with a carbon atom. Further examples includeanalogues in which a label is attached through a cleavable linker to the5-position of cytosine or thymine or to the 7-position of deaza-adenineor deaza-guanine. Other examples include analogues in which a smallchemical moiety such as —CH₂OCH₃ or —CH₂CH═CH₂ is used to cap the —OHgroup at the 3′-position of deoxyribose. Analogues of dideoxynucleotidescan similarly be prepared.

As used herein, a porous surface is a surface which contains pores or isotherwise uneven, such that the surface area of the porous surface isincreased relative to the surface area when the surface is smooth.

The present invention is directed to a method for sequencing a nucleicacid by detecting the identity of a nucleotide analogue after thenucleotide analogue is incorporated into a growing strand of DNA in apolymerase reaction, which comprises the following steps:

-   -   (i) attaching a 5′ end of the nucleic acid to a solid surface;    -   (ii) attaching a primer to the nucleic acid attached to the        solid surface;    -   (iii) adding a polymerase and one or more different nucleotide        analogues to the nucleic acid to thereby incorporate a        nucleotide analogue into the growing strand of DNA, wherein the        incorporated nucleotide analogue terminates the polymerase        reaction and wherein each different nucleotide analogue        comprises (a) a base selected from the group consisting of        adenine, guanine, cytosine, thymine, and uracil, and their        analogues; (b) a unique label attached through a cleavable        linker to the base or to an analogue of the base; (c) a        deoxyribose; and (d) a cleavable chemical group to cap an —OH        group at a 3′-position of the deoxyribose;    -   (iv) washing the solid surface to remove unincorporated        nucleotide analogues;    -   (v) detecting the unique label attached to the nucleotide        analogue that has been incorporated into the growing strand of        DNA, so as to thereby identify the incorporated nucleotide        analogue;    -   (vi) adding one or more chemical compounds to permanently cap        any unreacted —OH group on the primer attached to the nucleic        acid or on a primer extension strand formed by adding one or        more nucleotides or nucleotide analogues to the primer;    -   (vii) cleaving the cleavable linker between the nucleotide        analogue that was incorporated into the growing strand of DNA        and the unique label;    -   (viii) cleaving the cleavable chemical group capping the —OH        group at the 3′-position of the deoxyribose to uncap the —OH        group, and washing the solid surface to remove cleaved        compounds; and    -   (ix) repeating steps (iii) through (viii) so as to detect the        identity of a newly incorporated nucleotide analogue into the        growing strand of DNA;    -   wherein if the unique label is a dye, the order of steps (v)        through (vii) is: (v), (vi), and (vii); and    -   wherein if the unique label is a mass tag, the order of        steps (v) through (vii) is: (vi), (vii), and (v).

In one embodiment of any of the nucleotide analogues described herein,the nucleotide base is adenine. In one embodiment, the nucleotide baseis guanine. In one embodiment, the nucleotide base is cytosine. In oneembodiment, the nucleotide base is thymine. In one embodiment, thenucleotide base is uracil. In one embodiment, the nucleotide base is ananalogue of adenine. In one embodiment, the nucleotide base is ananalogue of guanine. In one embodiment, the nucleotide base is ananalogue of cytosine. In one embodiment, the nucleotide base is ananalogue of thymine. In one embodiment, the nucleotide base is ananalogue of uracil.

In different embodiments of any of the inventions described herein, thesolid surface is glass, silicon, or gold. In different embodiments, thesolid surface is a magnetic bead, a chip, a channel in a chip, or aporous channel in a chip. In one embodiment, the solid surface is glass.In one embodiment, the solid surface is silicon. In one embodiment, thesolid surface is gold. In one embodiments, the solid surface is amagnetic bead. In one embodiment, the solid surface is a chip. In oneembodiment, the solid surface is a channel in a chip. In one embodiment,the solid surface is a porous channel in a chip. Other materials canalso be used as long as the material does not interfere with the stepsof the method.

In one embodiment, the step of attaching the nucleic acid to the solidsurface comprises:

-   -   (i) coating the solid surface with a phosphine moiety,    -   (ii) attaching an azido group to the 5′ end of the nucleic acid,        and    -   (iii) immobilizing the 5′ end of the nucleic acid to the solid        surface through interaction between the phosphine moiety on the        solid surface and the azido group on the 5′ end of the nucleic        acid.

In one embodiment, the step of coating the solid surface with thephosphine moiety comprises:

-   -   (i) coating the surface with a primary amine, and    -   (ii) covalently coupling a N-hydroxysuccinimidyl ester of        triarylphosphine with the primary amine.

In one embodiment, the nucleic acid that is attached to the solidsurface is a single-stranded deoxyribonucleic acid (DNA). In anotherembodiment, the nucleic acid that is attached to the solid surface instep (i) is a double-stranded DNA, wherein only one strand is directlyattached to the solid surface, and wherein the strand that is notdirectly attached to the solid surface is removed by denaturing beforeproceeding to step (ii). In one embodiment, the nucleic acid that isattached to the solid surface is a ribonucleic acid (RNA), and thepolymerase in step (iii) is reverse transcriptase.

In one embodiment, the primer is attached to a 3′ end of the nucleicacid in step (ii), and the attached primer comprises a stable loop andan —OH group at a 3′-position of a deoxyribose capable of self-primingin the polymerase reaction. In one embodiment, the step of attaching theprimer to the nucleic acid comprises hybridizing the primer to thenucleic acid or ligating the primer to the nucleic acid. In oneembodiment, the primer is attached to the nucleic acid through aligation reaction which links the 3′ end of the nucleic acid with the 5′end of the primer.

In one embodiment, one or more of four different nucleotide analogs isadded in step (iii), wherein each different nucleotide analoguecomprises a different base selected from the group consisting of thymineor uracil or an analogue of thymine or uracil, adenine or an analogue ofadenine, cytosine or an analogue of cytosine, and guanine or an analogueof guanine, and wherein each of the four different nucleotide analoguescomprises a unique label.

In one embodiment, the cleavable chemical group that caps the —OH groupat the 3′-position of the deoxyribose in the nucleotide analogue is—CH₂OCH₃ or —CH₂CH═CH₂. Any chemical group could be used as long as thegroup 1) is stable during the polymerase reaction, 2) does not interferewith the recognition of the nucleotide analogue by polymerase as asubstrate, and 3) is cleavable.

In one embodiment, the unique label that is attached to the nucleotideanalogue is a fluorescent moiety or a fluorescent semiconductor crystal.In further embodiments, the fluorescent moiety is selected from thegroup consisting of 5-carboxyfluorescein, 6-carboxyrhodamine-6G,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine. Inone embodiment, the fluorescent moiety is 5-carboxyfluorescein. In oneembodiment, the fluorescent moiety is 6-carboxyrhodamine-6G,N,N,N′,N′-tetramethyl-6-carboxyrhodamine. In one embodiment, thefluorescent moiety is 6-carboxy-X-rhodamine.

In one embodiment, the unique label that is attached to the nucleotideanalogue is a fluorescence energy transfer tag which comprises an energytransfer donor and an energy transfer acceptor. In further embodiments,the energy transfer donor is 5-carboxyfluorescein or cyanine, andwherein the energy transfer acceptor is selected from the groupconsisting of dichlorocarboxyfluorescein,dichloro-6-carboxyrhodamine-6G,dichloro-N,N,N′,N′-tetramethyl-6-carboxyrhodamine, anddichloro-6-carboxy-X-rhodamine. In one embodiment, the energy transferacceptor is dichlorocarboxyfluorescein. In one embodiment, the energytransfer acceptor is dichloro-6-carboxyrhodamine-6G. In one embodiment,the energy transfer acceptor isdichloro-N,N,N′,N′-tetramethyl-6-carboxyrhodamine. In one embodiment,the energy transfer acceptor is dichloro-6-carboxy-X-rhodamine.

In one embodiment, the unique label that is attached to the nucleotideanalogue is a mass tag that can be detected and differentiated by a massspectrometer; In further embodiments, the mass tag is selected from thegroup consisting of a 2-nitro-α-methyl-benzyl group, a2-nitro-α-methyl-3-fluorobenzyl group, a2-nitro-α-methyl-3,4-difluorobenzyl group, and a2-nitro-α-methyl-3,4-dimethoxybenzyl group. In one embodiment, the masstag is a 2-nitro-α-methyl-benzyl group. In one embodiment, the mass tagis a 2-nitro-α-methyl-3-fluorobenzyl group. In one embodiment, the masstag is a 2-nitro-α-methyl-3,4-difluorobenzyl group. In one embodiment,the mass tag is a 2-nitro-α-methyl-3,4-dimethoxybenzyl group. In oneembodiment, the mass tag is detected using a parallel mass spectrometrysystem which comprises a plurality of atmospheric pressure chemicalionization mass spectrometers for parallel analysis of a plurality ofsamples comprising mass tags.

In one embodiment, the unique label is attached through a cleavablelinker to a 5-position of cytosine or thymine or to a 7-position ofdeaza-adenine or deaza-guanine. The unique label could also be attachedthrough a cleavable linker to another position in the nucleotideanalogue as long as the attachment of the label is stable during thepolymerase reaction and the nucleotide analog can be recognized bypolymerase as a substrate. For example, the cleavable label could beattached to the deoxyribose.

In one embodiment, the linker between the unique label and thenucleotide analogue is cleaved by a means selected from the groupconsisting of one or more of a physical means, a chemical means, aphysical chemical means, heat, and light. In one embodiment, the linkeris cleaved by a physical means. In one embodiment, the linker is cleavedby a chemical means. In one embodiment, the linker is cleaved by aphysical chemical means. In one embodiment, the linker is cleaved byheat. In one embodiment, the linker is cleaved by light. In oneembodiment, the linker is cleaved by ultraviolet light. In a furtherembodiment, the cleavable linker is a photocleavable linker whichcomprises a 2-nitrobenzyl moiety.

In one embodiment, the cleavable chemical group used to cap the —OHgroup at the 3′-position of the deoxyribose is cleaved by a meansselected from the group consisting of one or more of a physical means, achemical means, a physical chemical means, heat, and light. In oneembodiment, the linker is cleaved by a physical chemical means. In oneembodiment, the linker is cleaved by heat. In one embodiment, the linkeris cleaved by light. In one embodiment, the linker is cleaved byultraviolet light.

In one embodiment, the chemical compounds added in step (vi) topermanently cap any unreacted —OH group on the primer attached to thenucleic acid or on the primer extension strand are a polymerase and oneor more different dideoxynucleotides or analogues of dideoxynucleotides.In further embodiments, the different dideoxynucleotides are selectedfrom the group consisting of 2′,3′-dideoxyadenosine 5′-triphosphate,2′,3′-dideoxyguanosine 5′-triphosphate, 2′,3′-dideoxycytidine5′-triphosphate, 2′,3′-dideoxythymidine 5′-triphosphate,2′,3′-dideoxyuridine 5′-triphosphase, and their analogues. In oneembodiment, the dideoxynucleotide is 2′,3′-dideoxyadenosine5′-triphosphate. In one embodiment, the dideoxynucleotide is2′,3′-dideoxyguanosine 5′-triphosphate. In one embodiment, thedideoxynucleotide is 2′,3′-dideoxycytidine 5′-triphosphate. In oneembodiment, the dideoxynucleotide is 2′,3′-dideoxythymidine5′-triphosphate. In one embodiment, the dideoxynucleotide is2′,3′-dideoxyuridine 5′-triphosphase. In one embodiment, thedideoxynucleotide is an analogue of 2′,3′-dideoxyadenosine5′-triphosphate. In one embodiment, the dideoxynucleotide is an analogueof 2′,3′-dideoxyguanosine 5′-triphosphate. In one embodiment, thedideoxynucleotide is an analogue of 2′,3′-dideoxycytidine5′-triphosphate. In one embodiment, the dideoxynucleotide is an analogueof 2′,3′-dideoxythymidine 5′-triphosphate. In one embodiment, thedideoxynucleotide is an analogue of 2′,3′-dideoxyuridine5′-triphosphase.

In one embodiment, a polymerase and one or more of four differentdideoxynucleotides are added in step (vi), wherein each differentdideoxynucleotide is selected from the group consisting of2′,3′-dideoxyadenosine 5′-triphosphate or an analogue of2′,3′-dideoxyadenosine 5′-triphosphate; 2′,3′-dideoxyguanosine5′-triphosphate or an analogue of 2′,3′-dideoxyguanosine5′-triphosphate; 2′,3′-dideoxycytidine 5′-triphosphate or an analogue of2′,3′-dideoxycytidine 5′-triphosphate; and 2′,3′-dideoxythymidine5′-triphosphate or 2′,3′-dideoxyuridine 5′-triphosphase or an analogueof 2′,3′-dideoxythymidine 5′-triphosphate or an analogue of2′,3′-dideoxyuridine 5′-triphosphase. In one embodiment, thedideoxynucleotide is 2′,3′-dideoxyadenosine 5′-triphosphate. In oneembodiment, the dideoxynucleotide is an analogue of2′,3′-dideoxyadenosine 5′-triphosphate. In one embodiment, thedideoxynucleotide is 2′,3′-dideoxyguanosine 5′-triphosphate. In oneembodiment, the dideoxynucleotide is an analogue of2′,3′-dideoxyguanosine 5′-triphosphate. In one embodiment, thedideoxynucleotide is 2′,3′-dideoxycytidine 5′-triphosphate. In oneembodiment, the dideoxynucleotide is an analogue of2′,3′-dideoxycytidine 5′-triphosphate. In one embodiment, thedideoxynucleotide is 2′,3′-dideoxythymidine 5′-triphosphate. In oneembodiment, the dideoxynucleotide is 2′,3′-dideoxyuridine5′-triphosphase. In one embodiment, the dideoxynucleotide is an analogueof 2′,3′-dideoxythymidine 5′-triphosphate. In one embodiment, thedideoxynucleotide is an analogue of 2′,3′-dideoxyuridine5′-triphosphase.

Another type of chemical compound that reacts specifically with the —OHgroup could also be used to permanently cap any unreacted —OH group onthe primer attached to the nucleic acid or on an extension strand formedby adding one or more nucleotides or nucleotide analogues to the primer.

The invention provides a method for simultaneously sequencing aplurality of different nucleic acids, which comprises simultaneouslyapplying any of the methods disclosed herein for sequencing a nucleicacid to the plurality of different nucleic acids. In differentembodiments, the method can be used to sequence from one to over 100,000different nucleic acids simultaneously.

The invention provides for the use of any of the methods disclosedherein for detection of single nucleotide polymorphisms, geneticmutation analysis, serial analysis of gene expression, gene expressionanalysis, identification in forensics, genetic disease associationstudies, DNA sequencing, genomic sequencing, translational analysis, ortranscriptional analysis.

The invention provides a method of attaching a nucleic acid to a solidsurface which comprises:

-   -   (i) coating the solid surface with a phosphine moiety,    -   (ii) attaching an azido group to a 5′ end of the nucleic acid,        and    -   (iii) immobilizing the 5′ end of the nucleic acid to the solid        surface through interaction between the phosphine moiety on the        solid surface and the azido group on the 5′ end of the nucleic        acid.

In one embodiment, the step of coating the solid surface with thephosphine moiety comprises:

-   -   (i) coating the surface with a primary amine, and    -   (ii) covalently coupling a N-hydroxysuccinimidyl ester of        triarylphosphine with the primary amine.

In different embodiments, the solid surface is glass, silicon, or gold.In different embodiments, the solid surface is a magnetic bead, a chip,a channel in an chip, or a porous channel in a chip.

In different embodiments, the nucleic acid that is attached to the solidsurface is a single-stranded or double-stranded DNA or a RNA. In oneembodiment, the nucleic acid is a double-stranded DNA and only onestrand is attached to the solid surface. In a further embodiment, thestrand of the double-stranded DNA that is not attached to the solidsurface is removed by denaturing.

The invention provides for the use of any of the methods disclosedherein for attaching a nucleic acid to a surface for gene expressionanalysis, microarray based gene expression analysis, or mutationdetection, translational analysis, transcriptional analysis, or forother genetic applications.

The invention provides a nucleotide analogue which comprises:

-   -   (a) a base selected from the group consisting of adenine or an        analogue of adenine, cytosine or an analogue of cytosine,        guanine or an analogue of guanine, thymine or an analogue of        thymine, and uracil or an analogue of uracil;    -   (b) a unique label attached through a cleavable linker to the        base or to an analogue of the base;    -   (c) a deoxyribose; and    -   (d) a cleavable chemical group to cap an —OH group at a        3′-position of the deoxyribose.

In one embodiment of the nucleotide analogue, the cleavable chemicalgroup that caps the —OH group at the 3′-position of the deoxyribose is—CH₂OCH₃ or —CH₂CH═CH₂.

In one embodiment, the unique label is a fluorescent moiety or afluorescent semiconductor crystal. In further embodiments, thefluorescent moiety is selected from the group consisting of5-carboxyfluorescein, 6-carboxyrhodamine-6G,N,N,N′,N′-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine.

In one embodiment, the unique label is a fluorescence energy transfertag which comprises an energy transfer donor and an energy transferacceptor. In further embodiments, the energy, transfer donor is5-carboxyfluorescein or cyanine, and wherein the energy transferacceptor is selected from the group consisting ofdichlorocarboxyfluorescein, dichloro-6-carboxyrhodamine-6G,dichloro-N,N,N′,N′-tetramethyl-6-carboxyrhodamine, anddichloro-6-carboxy-X-rhodamine.

In one embodiment, the unique label is a mass tag that can be detectedand differentiated by a mass spectrometer. In further embodiments, themass tag is selected from the group consisting of a2-nitro-α-methyl-benzyl group, a 2-nitro-α-methyl-3-fluorobenzyl group,a 2-nitro-α-methyl-3,4-difluorobenzyl group, and a2-nitro-α-methyl-3,4-dimethoxybenzyl group.

In one embodiment, the unique label is attached through a cleavablelinker to a 5-position of cytosine or thymine or to a 7-position ofdeaza-adenine or deaza-guanine. The unique label could also be attachedthrough a cleavable linker to another position in the nucleotideanalogue as long as the attachment of the label is stable during thepolymerase reaction and the nucleotide analog can be recognized bypolymerase as a substrate. For example, the cleavable label could beattached to the deoxyribose.

In one embodiment, the linker between the unique label and thenucleotide analogue is cleavable by a means selected from the groupconsisting of one or more of a physical means, a chemical means, aphysical chemical means, heat, and light. In a further embodiment, thecleavable linker is a photocleavable linker which comprises a2-nitrobenzyl moiety.

In one embodiment, the cleavable chemical group used to cap the —OHgroup at the 3′-position of the deoxyribose is cleavable by a meansselected from the group consisting of one or more of a physical means, achemical means, a physical chemical means, heat, and light.

In different embodiments, the nucleotide analogue is selected from thegroup consisting of:

-   -   wherein Dye₁, Dye₂, Dye₃, and Dye₄ are four different unique        labels; and    -   wherein R is a cleavable chemical group used to cap the —OH        group at the 3′-position of the deoxyribose.

In different embodiments, the nucleotide analogue is selected from thegroup consisting of:

-   -   wherein R is —CH₂OCH₃ or —CH₂CH═CH₂.

In different embodiments, the nucleotide analogue is selected from thegroup consisting of:

-   -   wherein Tag₁, Tag₂, Tag₃, and Tag₄ are four different mass tag        labels; and    -   wherein R is a cleavable chemical group used to cap the —OH        group at the 3′-position of the deoxyribose.

In different embodiments, the nucleotide analogue is selected from thegroup consisting of:

-   -   wherein R is —CH₂OCH₃ or —CH₂CH═CH₂.

The invention provides for the use any of the nucleotide analoguesdisclosed herein for detection of single nucleotide polymorphisms,genetic mutation analysis, serial analysis of gene expression, geneexpression analysis, identification in forensics, genetic diseaseassociation studies, DNA sequencing, genomic sequencing, translationalanalysis, or transcriptional analysis.

The invention provides a parallel mass spectrometry system, whichcomprises a plurality of atmospheric pressure chemical ionization massspectrometers for parallel analysis of a plurality of samples comprisingmass tags. In one embodiment, the mass spectrometers are quadrupole massspectrometers. In one embodiment, the mass spectrometers aretime-of-flight mass spectrometers. In one embodiment, the massspectrometers are contained in one device. In one embodiment, the systemfurther comprises two turbo-pumps, wherein one pump is used to generatea vacuum and a second pump is used to remove undesired elements. In oneembodiment, the system comprises at least three mass spectrometers. Inone embodiment, the mass tags have molecular weights between 150 daltonsand 250 daltons. The invention provides for the use of the system forDNA sequencing analysis, detection of single nucleotide polymorphisms,genetic mutation analysis, serial analysis of gene expression, geneexpression analysis, identification in forensics, genetic diseaseassociation studies, DNA sequencing, genomic sequencing, translationalanalysis, or transcriptional analysis.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

1. The Sequencing by Synthesis Approach

Sequencing DNA by synthesis involves the detection of the identity ofeach nucleotide as it is incorporated into the growing strand of DNA inthe polymerase reaction. The fundamental requirements for such a systemto work are: (1) the availability of 4 nucleotide analogues (aA, aC, aG,aT) each labeled with a unique label and containing a chemical moietycapping the 3′-OH group; (2) the 4 nucleotide analogues (aA, aC, aG, aT)need to be efficiently and faithfully incorporated by DNA polymerase asterminators in the polymerase reaction; (3) the tag and the groupcapping the 3′-OH need to be removed with high yield to allow theincorporation and detection of the next nucleotide; and (4) the growingstrand of DNA should survive the washing, detection and cleavageprocesses to remain annealed to the DNA template.

The sequencing by synthesis approach disclosed herein is illustrated inFIG. 2A-2B. In FIG. 2A, an example is shown where the unique labels arefluorescent dyes and the surface is a chip; in FIG. 2B, the uniquelabels are mass tags and the surface is channels etched into a chip. Thesynthesis approach uses a solid surface such as a glass chip with animmobilized DNA template that is able to self prime for initiating thepolymerase reaction, and four nucleotide analogues(_(3′-RO)-A-_(LABEL1), _(3′-RO)-C-_(LABEL2), _(3′-RO)-G-_(LABEL3),_(3′—OR)-T-_(LABEL4)) each labeled with a unique label, e.g. afluorescent dye or a mass tag, at a specific location on the purine orpyrimidine base, and a small cleavable chemical group (R) to cap the3′-OH group. Upon adding the four nucleotide analogues and DNApolymerase, only one nucleotide analogue that is complementary to thenext nucleotide on the template is incorporated by the polymerase oneach spot of the surface (step 1 in FIGS. 2A and 2B).

As shown in FIG. 2A, where the unique labels are dyes, after removingthe excess reagents and washing away any unincorporated nucleotideanalogues on the chip, a detector is used to detect the unique label.For example, a four color fluorescence imager is used to image thesurface of the chip, and the unique fluorescence emission from aspecific dye on the nucleotide analogues on each spot of the chip willreveal the identity of the incorporated nucleotide (step 2 in FIG. 2A).After imaging, the small amount of unreacted 3′-OH group on theself-primed template moiety is capped by excess dideoxynucleosidetriphosphates (ddNTPs) (ddATP, ddGTP, ddTTP, and ddCTP) and DNApolymerase to avoid interference with the next round of synthesis (step3 in FIG. 2A), a concept similar to the capping step in automated solidphase DNA synthesis (Caruthers, 1985). The ddNTPs, which lack a3′-hydroxyl group, are chosen to cap the unreacted 3′-OH of thenucleotide due to their small size compared with the dye-labelednucleotides, and the excellent efficiency with which they areincorporated by DNA polymerase. The dye moiety is then cleaved by light(˜350 nm), and the R group protecting the 3′-OH is removed chemically togenerate free 3′-OH group with high yield (step 4 in FIG. 2A). A washingstep is applied to wash away the cleaved dyes and the R group. Theself-primed DNA moiety on the chip at this stage is ready for the nextcycle of the reaction to identify the next nucleotide sequence of thetemplate DNA (step 5 in FIG. 2A).

It is a routine procedure now to immobilize high density (>10,000 spotsper chip) single stranded DNA on a 4 cm×1 cm glass chip (Schena et al.1995). Thus, in the DNA sequencing system disclosed herein, more than10,000 bases can be identified after each cycle and after 100 cycles, amillion base pairs will be generated from one sequencing chip.

Possible DNA polymerases include Thermo Sequenase, Taq FS DNApolymerase, T7 DNA polymerase, and Vent (exo-) DNA polymerase. Thefluorescence emission from each specific dye can be detected using afluorimeter that is equipped with an accessory to detect fluorescencefrom a glass slide. For large scale evaluation, a multi-color scanningsystem capable of detecting multiple different fluorescent dyes (500nm-700 nm) (GSI Lumonics ScanArray 5000 Standard Biochip ScanningSystem) on a glass slide can be used.

An example of the sequencing by synthesis approach using mass tags isshown in FIG. 2B. The approach uses a solid surface, such as a poroussilica glass channels in a chip, with immobilized DNA template that isable to self prime for initiating the polymerase reaction, and fournucleotide analogues (_(3′-RO)-A-_(Tag1), _(3′-RO)-C-_(Tag2),_(3′-RO)-G-_(Tag3), _(3′-RO)-T-_(Tag4)) each labeled with a uniquephotocleavable mass tag on the specific location of the base, and asmall cleavable chemical group (R) to cap the 3′-OH group. Upon addingthe four nucleotide analogues and DNA polymerase, only one nucleotideanalogue that is complementary to the next nucleotide on the template isincorporated by polymerase in each channel of the glass chip (step 1 inFIG. 2B). After removing the excess reagents and washing away anyunincorporated nucleotide analogues on the chip, the small amount ofunreacted 3′-OH group on the self-primed template moiety is capped byexcess ddNTPs (ddATP, ddGTP, ddTTP and ddCTP) and DNA polymerase toavoid interference with the next round of synthesis (step 2 in FIG. 2B).The ddNTPs are chosen to cap the unreacted 3′-OH of the nucleotide dueto their small size compared with the labeled nucleotides, and theirexcellent efficiency to be incorporated by DNA polymerase. The mass tagsare cleaved by irradiation with light (˜350 nm) (step 3 in FIG. 2B) andthen detected with a mass spectrometer. The unique mass of each tagyields the identity of the nucleotide in each channel (step 4 in FIG.2B). The R protecting group is then removed chemically and washed awayto generate free 3′-OH group with high yield (step 5 in FIG. 2B). Theself-primed DNA moiety on the chip at this stage is ready for the nextcycle of the reaction to identify the next nucleotide sequence of thetemplate DNA (step 6 in FIG. 2B).

Since the development of new ionization techniques such as matrixassisted laser desorption ionization (MALDI) and electrospray ionization(ESI), mass spectrometry has become an indispensable tool in many areasof biomedical research. Though these ionization methods are suitable forthe analysis of bioorganic molecules, such as peptides and proteins,improvements in both detection and sample preparation are required forimplementation of mass spectrometry for DNA sequencing applications.Since the approach disclosed herein uses small and stable mass tags,there is no need to detect large DNA sequencing fragments directly andit is not necessary to use MALDI or ESI methods for detection.Atmospheric pressure chemical ionization (APCI) is an ionization methodthat uses a gas-phase ion-molecular reaction at atmospheric pressure(Dizidic et al. 1975). In this method, samples are introduced by eitherchromatography or flow injection into a pneumatic nebulizer where theyare converted into small droplets by a high-speed beam of nitrogen gas.When the heated gas and solution arrive at the reaction area, the excessamount of solvent is ionized by corona discharge. This ionized mobilephase acts as the ionizing agent toward the samples and yields pseudomolecular (M+H)⁺ and (M−H)⁻ ions. Due to the corona discharge ionizationmethod, high ionization efficiency is attainable, maintaining stableionization conditions with detection sensitivity lower than femtomoleregion for small and stable organic compounds. However, due to thelimited detection of large molecules, ESI and MALDI have replaced APCIfor analysis of peptides and nucleic acids. Since in the approachdisclosed the mass tags to be detected are relatively small and verystable organic molecules, the ability to detect large biologicalmolecules gained by using ESI and MALDI is not necessary. APCI hasseveral advantages over ESI and MALDI because it does not require anytedious sample preparation such as desalting or mixing with matrix toprepare crystals on a target plate. In ESI, the sample nature and samplepreparation conditions (i.e. the existence of buffer or inorganic salts)suppress the ionization efficiency. MALDI requires the addition ofmatrix prior to sample introduction into the mass spectrometer and itsspeed is often limited by the need to search for an ideal irradiationspot to obtain interpretable mass spectra. These limitations areovercome by APCI because the mass tag solution can be injected directlywith no additional sample purification or preparation into the massspectrometer. Since the mass tagged samples are volatile and have smallmass numbers, these compounds are easily detectable by APCI ionizationwith high sensitivity. This system can be scaled up into a highthroughput operation.

Each component of the sequencing by synthesis system is described inmore detail below.

2. Construction of a Surface Containing Immobilized Self-Primed DNAMoiety

The single stranded DNA template immobilized on a surface is preparedaccording to the scheme shown in FIG. 3. The surface can be, forexample, a glass chip, such as a 4 cm×1 cm glass chip, or channels in aglass chip. The surface is first treated with 0.5 M NaOH, washed withwater, and then coated with high density 3-aminopropyltrimethoxysilanein aqueous ethanol (Woolley et al. 1994) forming a primary aminesurface. N-Hydroxy Succinimidyl (NHS) ester of triarylphosphine (1) iscovalently coupled with the primary amine group converting the aminesurface to a novel triarylphosphine surface, which specifically reactswith DNA containing an azido group (2) forming a chip with immobilizedDNA. Since the azido group is only located at the 5′ end of the DNA andthe coupling reaction is through the unique reaction of thetriarylphosphine moiety with the azido group in aqueous solution (Saxonand Bertozzi 2000), such a DNA surface will provide an optimal conditionfor hybridization.

The NHS ester of triarylphosphine (1) is prepared according to thescheme shown in FIG. 4. 3-diphenylphosphino-4-methoxycarbonyl-benzoicacid (3) is prepared according to the procedure described by Bertozzi etal. (Saxon and Bertozzi 2000). Treatment of (3) withN-Hydroxysuccinimide forms the corresponding NHS ester (4). Coupling of(4) with an amino carboxylic acid moiety produces compound (5) that hasa long linker (n=1 to 10) for optimized coupling with DNA on thesurface. Treatment of (5) with N-Hydroxysuccinimide generates the NHSester (1) which is ready for coupling with the primary amine coatedsurface (FIG. 3).

The azido labeled DNA (2) is synthesized according to the scheme shownin FIG. 5. Treatment of ethyl ester of 5-bromovaleric acid with sodiumazide and then hydrolysis produces 5-azidovaleric acid (Khoukhi et al.,1987), which is subsequently converted to a NHS ester for coupling withan amino linker modified oligonucleotide primer. Using the azido-labeledprimer to perform polymerase chain reaction (PCR) reaction generatesazido-labeled DNA template (2) for coupling with thetriarylphosphine-modified surface (FIG. 3).

The self-primed DNA template moiety on the sequencing chip isconstructed as shown in FIGS. 6(A & B) using enzymatic ligation. A5′-phosphorylated, 3′-OH capped loop oligonucleotide primer (B) issynthesized by a solid phase DNA synthesizer. Primer (B) is synthesizedusing a modified C phosphoramidite whose 3′-OH is capped with either aMOM (—CH₂OCH₃) group or an allyl (—CH₂CH═CH₂) group (designated by “R”in FIG. 6) at the 3′-end of the oligonucleotide to prevent the selfligation of the primer in the ligation reaction. Thus, the looped primercan only ligate to the 3′-end of the DNA templates that are immobilizedon the sequencing chip using T4 RNA ligase (Zhang et al. 1996) to formthe self-primed DNA template moiety (A). The looped primer (B) isdesigned to contain a very stable loop (Antao et al. 1991) and a stemcontaining the sequence of M13 reverse DNA sequencing primer forefficient priming in the polymerase reaction once the primer is ligatedto the immobilized DNA on the sequencing chip and the 3′-OH cap group ischemically cleaved off (Ireland et al. 1986; Kamal et al. 1999).

3. Sequencing by Synthesis Evaluation Using Nucleotide Analogues_(3′-HO)-A-_(Dye1), _(3′-HO)-C-_(Dye2), _(3′-HO)-G-_(Dye3),_(3′-HO)-T-_(Dye4)

A scheme has been developed for evaluating the photocleavage efficiencyusing different dyes and testing the sequencing by synthesis approach.Four nucleotide analogues _(3′-HO)-A-_(Dye1), _(3′-HO)-C-_(Dye2),_(3′-HO)-G-_(Dye3), _(3′-HO)-T=_(Dye4) each labeled with a uniquefluorescent dye through a photocleavable linker are synthesized and usedin the sequencing by synthesis approach. Examples of dyes include, butare not limited to: Dye1=FAM, 5-carboxyfluorescein; Dye2=R6G,6-carboxyrhodamine-6G; Dye3=TAM,N,N,N′,N′-tetramethyl-6-carboxyrhodamine; and Dye4=ROX,6-carboxy-X-rhodamine. The structures of the 4 nucleotide analogues areshown in FIG. 7 (R═H).

The photocleavable 2-nitrobenzyl moiety has been used to link biotin toDNA and protein for efficient removal by UV light (˜350 nm) (Olejnik etal. 1995, 1999). In the approach disclosed herein the 2-nitrobenzylgroup is used to bridge the fluorescent dye and nucleotide together toform the dye labeled nucleotides as shown in FIG. 7.

As a representative example, the synthesis of _(3′-HO)-G-_(Dye3)(Dye3=Tam) is shown in FIG. 8. 7-deaza-alkynylamino-dGTP is preparedusing well-established procedures (Prober et al. 1987; Lee et al. 1992and Hobbs et al. 1991). Linker-Tam is synthesized by coupling thePhotocleavable Linker (Rollaf 1982) with NHS-Tam.7-deaza-alkynylamino-dGTP is then coupled with the Linker-Tam to produce_(3′-HO)-G-_(TAM). The nucleotide analogues with a free 3′-OH (i.e.,R═H) are good substrates for the polymerase. An immobilized DNA templateis synthesized (FIG. 9) that contains a portion of nucleotide sequenceACGTACGACGT (SEQ ID NO: 1) that has no repeated sequences after thepriming site. _(3′-HO)-A-_(Dye1) and DNA polymerase are added to theself-primed DNA moiety and it is incorporated to the 3′ site of the DNA.Then the steps in FIG. 2A are followed (the chemical cleavage step isnot required here because the 3′-OH is free) to detect the fluorescentsignal from Dye-1 at 520 nm. Next, _(3′-HO)-C-_(Dye2) is added to imagethe fluorescent signal from Dye-2 at 550 nm. Next, _(3′-HO)-G-_(Dye3) isadded to image the fluorescent signal from Dye-3 at 580 nm, and finally_(3′-HO)-T-_(Dye4) is added to image the fluorescent signal from Dye-4at 610 nm.

Results on Photochemical Cleavage Efficiency

The expected photolysis products of DNA containing a photocleavablefluorescent dye at the 3′ end of the DNA are shown in FIG. 10. The2-nitrobenzyl moiety has been successfully employed in a wide range ofstudies as a photocleavable-protecting group (Pillai 1980). Theefficiency of the photocleavage step depends on several factorsincluding the efficiency of light absorption by the 2-nitrobenzylmoiety, the efficiency of the primary photochemical step, and theefficiency of the secondary thermal processes which lead to the finalcleavage process (Turro 1991). Burgess et al. (1997) have reported thesuccessful photocleavage of a fluorescent dye attached through a2-nitrobenzyl linker on a nucleotide moiety, which shows that thefluorescent dye is not quenching the photocleavage process. Aphotoliable protecting group based on the 2-nitrobenzyl chromophore hasalso been developed for biological labeling applications that involvephotocleavage (Olejnik et al. 1999). The protocol disclosed herein isused to optimize the photocleavage process shown in FIG. 10. Theabsorption spectra of 2-nitro benzyl compounds are examined and comparedquantitatively to the absorption spectra of the fluorescent dyes. Sincethere will be a one-to-one relationship between the number of2-nitrobenzyl moieties and the dye molecules, the ratio of extinctioncoefficients of these two species will reflect the competition for lightabsorption at specific wavelengths. From this information, thewavelengths at which the 2-nitrobenzyl moieties absorbed mostcompetitively can be determined, similar to the approach reported byOlejnik et al. (1995).

A photolysis setup can be used which allows a high throughput ofmonochromatic light from a 1000 watt high pressure xenon lamp (LX1000UV,ILC) in conjunction with a monochromator (Kratos, SchoeffelInstruments). This instrument allows the evaluation of the photocleavageof model systems as a function of the intensity and excitationwavelength of the absorbed light. Standard analytical analysis is usedto determine the extent of photocleavage. From this information, theefficiency of the photocleavage as a function of wavelength can bedetermined. The wavelength at which photocleavage occurs mostefficiently can be selected as for use in the sequencing system.

Photocleavage results have been obtained using a model system as shownin FIG. 11. Coupling of PC-LC-Biotin-NHS ester (Pierce, Rockford, Ill.)with 5-(aminoacetamido)-fluorescein (5-aminoFAM) (Molecular Probes,Eugene, Oreg.) in dimethylsulfonyl oxide (DMSO)/NaHCO₃ (pH=8.2)overnight at room temperature produces PC-LC-Biotin-FAM which iscomposed of a biotin at one end, a photocleavable 2-nitrobenzyl group inthe middle, and a dye tag (FAM) at the other end. This photocleavablemoiety closely mimics the designed photocleavable nucleotide analoguesshown in FIG. 10. Thus the successful photolysis of the PC-LC-Biotin-FAMmoiety provides proof of the principle of high efficiency photolysis asused in the DNA sequencing system. For photolysis study,PC-LC-Biotin-FAM is first immobilized on a microscope glass slide coatedwith streptavidin (XENOPORE, Hawthorne, N.J.). After washing off thenon-immobilized PC-LC-Biotin-FAM, the fluorescence emission spectrum ofthe immobilized PC-LC-Biotin-FAM was taken as shown in FIG. 12 (Spectruma). The strong fluorescence emission indicates that PC-LC-Biotin-FAM issuccessfully immobilized to the streptavidin coated slide surface. Thephotocleavability of the 2-nitrobenzyl linker by irradiation at 350 nmwas then tested. After 10 minutes of photolysis (λ_(irr)=350 nm; ˜0.5mW/cm²) and before any washing, the fluorescence emission spectrum ofthe same spot on the slide was taken that showed no decrease inintensity (FIG. 12, Spectrum b), indicating that the dye (FAM) was notbleached during the photolysis process at 350 nm. After washing theglass slide with HPLC water following photolysis, the fluorescenceemission spectrum of the same spot on the slide showed significantintensity decrease (FIG. 12, Spectrum c) which indicates that most ofthe fluorescence dye (FAM) was cleaved from the immobilized biotinmoiety and was removed by the washing procedure. This experiment showsthat high efficiency cleavage of the fluorescent dye can be obtainedusing the 2-nitrobenzyl photocleavable linker.

4. Sequencing by Synthesis Evaluation Using Nucleotide Analogues_(3′-RO)-A-_(Dye1), _(3′-RO)-C-_(Dye2), _(3′-RO)-G-_(Dye3),_(3′-RO)-T-_(Dye4)

Once the steps and conditions in Section 3 are optimized, the synthesisof nucleotide analogues _(3′-RO)-A-_(Dye1), _(3′-RO)-C-_(Dye2),_(3′-RO)-G-_(Dye3), _(3′-RO)-T-_(Dye4) can be pursued for further studyof the system. Here the 3′-OH is capped in all four nucleotideanalogues, which then can be mixed together with DNA polymerase and usedto evaluate the sequencing system using the scheme in FIG. 9. The MOM(—CH₂OCH₃) or allyl (—CH₂CH═CH₂) group is used to cap the 3′-OH groupusing well-established synthetic procedures (FIG. 13) (Fuji et al. 1975,Metzker et al. 1994). These groups can be removed chemically with highyield as shown in FIG. 14 (Ireland, et al. 1986; Kamal et al. 1999). Thechemical cleavage of the MOM and allyl groups is fairly mild andspecific, so as not to degrade the DNA template moiety. For example, thecleavage of the allyl group takes 3 minutes with more than 93% yield(Kamal et al. 1999), while the MOM group is reported to be cleaved withclose to 100% yield (Ireland, et al. 1986).

5. Using Energy Transfer Coupled Dyes to Optimize the Sequencing bySynthesis System

The spectral property of the fluorescent tags can be optimized by usingenergy transfer (ET) coupled dyes. The ET primer and ETdideoxynucleotides have been shown to be a superior set of reagents for4-color DNA sequencing that allows the use of one laser to excitemultiple sets of fluorescent tags (Ju et al. 1995). It has been shownthat DNA polymerase (Thermo Sequenase and Taq FS) can efficientlyincorporate the ET dye labeled dideoxynucleotides (Rosenblum et al.1997). These ET dye-labeled sequencing reagents are now widely used inlarge scale DNA sequencing projects, such as the human genome project. Alibrary of ET dye labeled nucleotide analogues can be synthesized asshown in FIG. 15 for optimization of the DNA sequencing system. The ETdye set (FAM-Cl₂FAM, FAM-Cl₂R6G, FAM-Cl₂TAM, FAM-Cl₂ROX) using FAM as adonor and dichloro(FAM, R6G, TAM, ROX) as acceptors has been reported inthe literature (Lee et al. 1997) and constitutes a set of commerciallyavailable DNA sequencing reagents. These ET dye sets have been proven toproduce enhanced fluorescence intensity, and the nucleotides labeledwith these ET dyes at the 5-position of T and C and the 7-position of Gand A are excellent substrates of DNA polymerase. Alternatively, an ETdye set can be constructed using cyanine (Cy2) as a donor and Cl₂FAM,Cl₂R6G, Cl₂TAM, or Cl₂ROX as energy acceptors. Since Cy2 possesseshigher molar absorbance compared with the rhodamine and fluoresceinderivatives, an ET system using Cy2 as a donor produces much strongerfluorescence signals than the system using FAM as a donor (Hung et al.1996). FIG. 16 shows a synthetic scheme for an ET dye labeled nucleotideanalogue with Cy2 as a donor and Cl₂FAM as an acceptor using similarcoupling chemistry as for the synthesis of an energy transfer systemusing FAM as a donor (Lee et al. 1997). Coupling of Cl₂FAM (I) withspacer 4-aminomethylbenzoic acid (II) produces III, which is thenconverted to NHS ester IV. Coupling of IV with amino-Cy2, and thenconverting the resulting compound to a NHS ester produces V, whichsubsequently couples with amino-photolinker nucleotide VI yields the ETdye labeled nucleotide VII.

6. Sequencing by Synthesis Evaluation Using Nucleotide Analogues_(3′-HO)-A-_(Tag1), _(3′-HO)-C-_(Tag2), _(3′-HO)-G-_(Tag3),_(3′-HO)-T-_(Tag4)

The precursors of four examples of mass tags are shown in FIG. 17. Theprecursors are: (a) acetophenone; (b) 3-fluoroacetophenone; (c)3,4-difluoroacetophenone; and (d) 3,4-dimethoxyacetophenone. Uponnitration and reduction, four photoactive tags are produced from thefour precursors and used to code for the identity of each of the fournucleotides (A, C, G, T). Clean APCI mass spectra are obtained for thefour mass tag precursors (a, b, c, d) as shown in FIG. 18. The peak withm/z of 121 is a, 139 is b, 157 is c, and 181 is d. This result showsthat these four mass tags are extremely stable and produce very highresolution data in an APCI mass spectrometer with no cross talk betweenthe mass tags. In the examples shown below, each of the unique m/z fromeach mass tag translates to the identity of the nucleotide [Tag-1 (m/z,150)=A; Tag-2 (m/z, 168)=C; Tag-3 (m/z, 186)=G; Tag-4 (m/z, 210)=T].

Different combinations of mass tags and nucleotides can be used, asindicated by the general scheme: _(3′-HO)-A-_(Tag1), _(3′-HO)-C-_(Tag2),_(3′-HO)-G-_(Tag3), _(3′-HO)-T-_(Tag4) where Tag1, Tag2, Tag3, and Tag4are four different unique cleavable mass tags. Four specific examples ofnucleotide analogues are shown in FIG. 19. In FIG. 19, “R” is H when the3′-OH group is not capped. As discussed above, the photo cleavable2-nitro benzyl moiety has been used to link biotin to DNA and proteinfor efficient removal by UV light (˜350 nm) irradiation (Olejnik et al.1995, 1999). Four different 2-nitro benzyl groups with differentmolecular weights as mass tags are used to form the mass tag labelednucleotides as shown in FIG. 19: 2-nitro-α-methyl-benzyl (Tag-1) codesfor A; 2-nitro-α-methyl-3-fluorobenzyl (Tag-2) codes for C;2-nitro-α-methyl-3,4-difluorobenzyl (Tag-3) codes for G;2-nitro-α-methyl-3,4-dimethoxybenzyl (Tag-4) codes for T.

As a representative example, the synthesis of the NHS ester of one masstag (Tag-3) is shown in FIG. 20. A similar scheme is used to create theother mass tags. The synthesis of _(3′-HO)-G-_(Tag3) is shown in FIG. 21using well-established procedures (Prober et al. 1987; Lee et al. 1992and Hobbs et al. 1991). 7-propargylamino-dGTP is first prepared byreacting 7-I-dGTP with N-trifluoroacetylpropargyl amine, which is thencoupled with the NHS-Tag-3 to produce _(3′-HO)-G-_(Tag3). The nucleotideanalogues with a free 3′-OH are good substrates for the polymerase.

The sequencing by synthesis approach can be tested using mass tags usinga scheme similar to that show for dyes in FIG. 9. A DNA templatecontaining a portion of nucleotide sequence that has no repeatedsequences after the priming site, is synthesized and immobilized to aglass channel. _(3′-HO)-A-_(Tag1) and DNA polymerase are added to theself-primed DNA moiety to allow the incorporation of the nucleotide intothe 3′ site of the DNA. Then the steps in FIG. 2B are followed (thechemical cleavage is not required here because the 3′-OH is free) todetect the mass tag from Tag-1 (m/z=150). Next, _(3′-HO)-C-_(Tag2) isadded and the resulting mass spectra is measured after cleaving Tag-2(m/z=168). Next, _(3′-HO)-G-_(Tag3) and _(3′-HO)-T-_(Tag4) are added inturn and the mass spectra of the cleavage products Tag-3 (m/z=186) andTag-4 (m/z=210) are measured. Examples of expected photocleavageproducts are shown in FIG. 22. The photocleavage mechanism is asdescribed above for the case where the unique labels are dyes. Lightabsorption (300-360 nm) by the aromatic 2-nitro benzyl moiety causesreduction of the 2-nitro group to a nitroso group and an oxygeninsertion into the carbon-hydrogen bond located in the 2-positionfollowed by cleavage and decarboxylation (Pillai 1980).

The synthesis of nucleotide analogues _(3′-RO)-A-_(Tag1),_(3′-RO)-C-_(Tag2), _(3′-RO)-G-_(Tag3), _(3′-RO)-T-_(Tag4) can bepursued for further study of the system a discussed above for the casewhere the unique labels are dyes. Here the 3′-OH is capped in all fournucleotide analogues, which then can be mixed together with DNApolymerase and used to evaluate the sequencing system using a schemesimilar to that in FIG. 9. The MOM (—CH₂OCH₃) or allyl (—CH₂CH═CH₂)group is used to cap the 3′-OH group using well-established syntheticprocedures (FIG. 13) (Fuji et al. 1975, Metzker et al. 1994). Thesegroups can be removed chemically with high yield as shown in FIG. 14(Ireland, et al. 1986; Kamal et al. 1999). The chemical cleavage of theMOM and allyl groups is fairly mild and specific, so as not to degradethe DNA template moiety.

7. Parallel Channel System for Sequencing by Synthesis

FIG. 23 illustrates an example of a parallel channel system. The systemcan be used with mass tag labels as shown and also with dye labels. Aplurality of channels in a silica glass chip are connected on each endof the channel to a well in a well plate. In the example shown there are96 channels each connected to its own wells. The sequencing system alsopermits a number of channels other than 96 to be used. 96 channeldevices for separating DNA sequencing and sizing fragments have beenreported (Woolley and Mathies 1994, Woolley et al. 1997, Simpson et al.1998). The chip is made by photolithographic masking and chemicaletching techniques. The photolithographically defined channel patternsare etched in a silica glass substrate, and then capillary channels (id˜100 μm) are formed by thermally bonding the etched substrate to asecond silica glass slide. Channels are porous to increase surface area.The immobilized single stranded DNA template chip is prepared accordingto the scheme shown in FIG. 3. Each channel is first treated with 0.5 MNaOH, washed with water, and is then coated with high density3-aminopropyltrimethoxysilane in aqueous ethanol (Woolley et al. 1994)forming a primary amine surface. Succinimidyl (NHS) ester oftriarylphosphine (1) is covalently coupled with the primary amine groupconverting the amine surface to a novel triarylphosphine surface, whichspecifically reacts with DNA containing an azido group (2) forming achip with immobilized DNA. Since the azido group is only located at the5′ end of the DNA and the coupling reaction is through the uniquereaction of triarylphosphine moiety with azido group in aqueous solution(Saxon and Bertozzi 2000), such a DNA surface provides an optimizedcondition for hybridization. Fluids, such as sequencing reagents andwashing solutions, can be easily pressure driven between the two 96 wellplates to wash and add reagents to each channel in the chip for carryingout the polymerase reaction as well as collecting the photocleavedlabels. The silica chip is transparent to ultraviolet light (λ˜350 nm).In the Figure, photocleaved mass tags are detected by an APCI massspectrometer upon irradiation with a UV light source.

8. Parallel Mass Tag Sequencing by Synthesis System

The approach disclosed herein comprises detecting four uniquephotoreleased mass tags, which can have molecular weights from 150 to250 daltons, to decode the DNA sequence, thereby obviating the issue ofdetecting large DNA fragments using a mass spectrometer as well as thestringent sample requirement for using mass spectrometry to directlydetect long DNA fragments. It takes 10 seconds or less to analyze eachmass tag using the APCI mass spectrometer. With 8 miniaturized APCI massspectrometers in a system, close to 100,000 bp of high quality digitalDNA sequencing data could be generated each day by each instrument usingthis approach. Since there is no separation and purificationrequirements using this approach, such a system is cost effective.

To make mass spectrometry competitive with a 96 capillary array methodfor analyzing DNA, a parallel mass spectrometer approach is needed. Sucha complete system has not been reported mainly due to the fact that mostof the mass spectrometers are designed to achieve adequate resolutionfor large biomolecules. The system disclosed herein requires thedetection of four mass tags, with molecular weight range between 150 and250 daltons, coding for the identity of the four nucleotides (A, C, G,T). Since a mass spectrometer dedicated to detection of these mass tagsonly requires high resolution for the mass range of 150 to 250 daltonsinstead of covering a wide mass range, the mass spectrometer can beminiaturized and have a simple design. Either quadrupole (including iontrap detector) or time-of-flight mass spectrometers can be selected forthe ion optics. While modern mass spectrometer technology has made itpossible to produce miniaturized mass spectrometers, most currentresearch has focused on the design of a single stand-alone miniaturizedmass spectrometer. Individual components of the mass spectrometer hasbeen miniaturized for enhancing the mass spectrometer analysiscapability (Liu et al. 2000, Zhang et al. 1999). A miniaturized massspectrometry system using multiple analyzers (up to 10) in parallel hasbeen reported (Badman and Cooks 2000). However, the mass spectrometer ofBadman and Cook was designed to measure only single samples rather thanmultiple samples in parallel. They also noted that the miniaturizationof the ion trap limited the capability of the mass spectrometer to scanwide mass ranges. Since the approach disclosed herein focuses ondetecting four small stable mass tags (the mass range is less than 300daltons), multiple miniaturized APCI mass spectrometers are easilyconstructed and assembled into a single unit for parallel analysis ofthe mass tags for DNA sequencing analysis.

A complete parallel mass spectrometry system includes multiple APCIsources interfaced with multiple analyzers, coupled with appropriateelectronics and power supply configuration. A mass spectrometry systemwith parallel detection capability will overcome the throughputbottleneck issue for application in DNA analysis. A parallel systemcontaining multiple mass spectrometers in a single device is illustratedin FIGS. 23 and 24. The examples in the figures show a system with threemass spectrometers in parallel. Higher throughput is obtained using agreater number of in parallel mass spectrometers.

As illustrated in FIG. 24, the three miniature mass spectrometers arecontained in one device with two turbo-pumps. Samples are injected intothe ion source where they are mixed with a nebulizer gas and ionized.One turbo pump is used as a differential pumping system to continuouslysweep away free radicals, neutral compounds and other undesirableelements coming from the ion source at the orifice between the ionsource and the analyzer. The second turbo pump is used to generate acontinuous vacuum in all three analyzers and detectors simultaneously.Since the corona discharge mode and scanning mode of mass spectrometersare the same for each miniaturized mass spectrometer, one power supplyfor each analyzer and the ionization source can provide the necessarypower for all three instruments. One power supply for each of the threeindependent detectors is used for spectrum collection. The data obtainedare transferred to three independent A/D converters and processed by thedata system simultaneously to identify the mass tag in the injectedsample and thus identify the nucleotide. Despite containing three massspectrometers, the entire device is able to fit on a laboratory benchtop.

9. Validate the Complete Sequencing by Synthesis System by SequencingP53 Genes

The tumor suppressor gene p53 can be used as a model system to validatethe DNA sequencing system. The p53 gene is one of the most frequentlymutated genes in human cancer (O'Connor et al. 1997). First, a base pairDNA template (shown below) is synthesized containing an azido group atthe 5′ end and a portion of the sequences from exon 7 and exon 8 of thep53 gene:

(SEQ ID NO: 2) 5′-N 3-TTCCTGCATGGGCG G CA T GAACC C GAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGG G ACGGAACAGCTTTG AGGTGC A TT-3′.

This template is chosen to explore the use of the sequencing system forthe detection of clustered hot spot single base mutations. Thepotentially mutated bases are underlined (A, G, C and T) in thesynthetic template. The synthetic template is immobilized on asequencing chip or glass channels, then the loop primer is ligated tothe immobilized template as described in FIG. 6, and then the steps inFIG. 2 are followed for sequencing evaluation. DNA templates generatedby PCR can be used to further validate the DNA sequencing system. Thesequencing templates can be generated by PCR using flanking primers (oneof the pair is labeled with an azido group at the 5′ end) in the intronregion located at each p53 exon boundary from a pool of genomic DNA(Boehringer, Indianapolis, Ind.) as described by Fu et al. (1998) andthen immobilized on the DNA chip for sequencing evaluation.

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1. A method of determining the identity of a nucleotide analogueincorporated into a nucleic acid primer extension strand, comprising: a)contacting a nucleic acid template attached to a solid surface with anucleic acid primer which hybridizes to the template; b) simultaneouslycontacting the product of step a) with a polymerase and four nucleotideanalogues which are either (i) aA, aC, aG, and aT, or (ii) aA, aC, aG,and aU, so as to incorporate one of the nucleotide analogues onto thenucleic acid primer and form a nucleic acid primer extension strand,wherein each nucleotide analogue within (i) or (ii) comprises a baselabeled with a unique label and contains a small removable chemicalmoiety capping the 3′-OH group of the sugar of the nucleotide analogue,wherein said small cleavable chemical group does not interfere with therecognition of the nucleotide analogue by polymerase as a substrate; andc) detecting the unique label of the incorporated nucleotide analogue,so as to thereby determine the identity of the nucleotide analogueincorporated into the nucleic acid primer extension strand.
 2. Themethod of claim 1, further comprising removing the chemical moietycapping the 3′-OH group of the sugar of the incorporated nucleotideanalogue, thereby permitting the incorporation of a further nucleotideanalogue so as to create a growing annealed nucleic acid primerextension strand.
 3. The method of claim 1, wherein each of the fourunique labels is a fluorescent label.
 4. The method of claim 3, whereinone of said four unique labels comprises ROX.
 5. The method of claim 3,wherein one of said four unique labels comprises R6G.
 6. The method ofclaim 1, wherein said base of at least one of said nucleotide analoguesis a deazapurine.